Directed cell migration toward chemoattractants, termed chemotaxis, is central to many physiologic events such as axon guidance, wound healing, and tissue morphogenesis. Inappropriate chemotaxis is a key feature of many human diseases, including tumor metastasis, asthma, arthritis, and atherosclerosis. Understanding the mechanisms of chemotaxis is therefore vital for understanding these chemotaxis-related diseases. The long- term goal of our research is to reveal how cells sense their chemical environment and control their migratory behaviors. Using Dictyostelium amoebae as our discovery tool and human cells as our translational tool, we focus on the potent intracellular signal phosphatidylinositol-3,4,5-triphosphate (PIP3), which is produced at the leading edge of cells and reorganizes the actin cytoskeleton. An important, but unanswered, question in the field of chemotaxis is how cells stably maintain the signaling network and remodel the actin cytoskeleton in chemoattractant gradients. To address this fundamental problem, our current studies identified: i) a signaling step that stabilizes directional sensing by persistently orientig Ras activation and PIP3 production, ii) a molecular link that directly binds to both PIP3 and the actin cytoskeleton, and iii) a conserved process in Dictyostelium and humans that turns off PIP3 signaling through translocation of the PIP3 phosphatase PTEN to the plasma membrane. In the next funding period, we propose to study each of these regulatory events and the mechanisms by which chemotactic signaling controls cell migration with high precision. In Aim 1, we will determine how directional sensing is spatially directed toward chemoattractants. We hypothesize that the activation of Ras GTPases and PIP3 production that occurs at the leading portion of cells is regulated by active Rho GTPases located at the rear end through chemical gradients. We will examine how Rho GTPases transmit signal to Ras GTPases. In Aim 2, we will determine how PIP3-binding monomeric myosin I converts the PIP3 signal to the actin cytoskeleton. We will test three models for the function of myosin I in cytoskeletal remodeling: connecting actin filaments to the plasma membrane, directly polymerizing actin, and recruiting actin nucleation factors. In Aim 3, we will delineate how human PTEN is recruited to the plasma membrane. We hypothesize that previously unidentified PTEN receptors in the plasma membrane mediate this process in human cells. We will examine the function of newly identified human PTEN-binding proteins in the localization of PTEN. Moreover, we will further determine the functional importance of the receptors in PIP3 signaling. The outcomes of our research are expected to provide a conceptual breakthrough into two central events in chemotaxis, directional sensing and cytoskeletal rearrangements, and may lead to development of chemotaxis-based treatments for cancer and inflammation.